In liquid chromatography, a fluidic sample and an eluent (liquid mobile phase) may be pumped through conduits and a column in which separation of sample components takes place. The column may comprise a material which is capable of separating different components of the fluidic analyte. Such a packing material, so-called beads which may comprise silica gel, may be filled into a column tube which may be connected to other elements (like a control unit, containers including sample and/or buffers) by conduits. The composition of the mobile phase can be adjusted by composing the mobile phase from different fluidic components with variable contributions.
Two-dimensional separation of a fluidic sample denotes a separation technique in which a first separation procedure in a first separation unit is performed to separate a fluidic sample into a plurality of fractions, and in which a subsequent second separation procedure in a second separation unit is performed to further separate the plurality of fractions into sub-fractions. Two-dimensional liquid chromatography (2D LC) may combine two liquid chromatography separation techniques.
Pavel Jandera, Tomas Hajek, Petr Cesla, “Comparison of various second-dimension gradient types in comprehensive two-dimensional liquid chromatography”, J. Sep. Sci. 2010, 33, 1382-1397, discloses that gradient elution provides significant improvement in peak capacity with respect to isocratic conditions. In the second dimension, gradients are limited to a short-time period available for separation. Various types of second-dimension gradients in comprehensive LC LC are compared: (i) “full in fraction”, (ii) “segment in fraction” and (iii) “continuously shifting” gradients, applied in orthogonal LC LC separations of phenolic acids and flavones on a polyethylene glycol column in the first dimension and two types of porous shell fused-core C18 columns in the second dimension (Ascentis Express and Kinetex). The porous shell columns provide narrow bandwidths and fast second-dimension separations at moderate operating pressure that allows important savings of the overall separation time in comprehensive LC LC separations. The effects of the gradient type on the bandwidths, theoretical peak capacity, separation time and column pressure in the second dimension were investigated. The type of gradient program controls the range of lipophilicity of sample compounds that can be separated in the second-dimension reversed-phase time period. This range can be calibrated using alkylbenzene standards, to design the separation conditions for complete sample separation, avoiding harmful wrap around of non-eluted compounds to the subsequent second-dimension fractions.
However, such a concept of two-dimensional liquid chromatography is cumbersome for a user since the second dimension is divided into a plurality of completely unrelated gradient runs, involving the need for a user to program each of these gradient runs individually.
Furthermore, when plotting the result of a 2D LC measurement in a two-dimensional coordinate system, it may happen that relatively large area regions may remain empty or basically empty. This corresponds to the fact that a certain portion of the measurement time is spent inefficiently.